3.1. Pomegranate and Sumac Extracts
Extracts obtained from pomegranate (Punica granatum
L.) peels and from sumac (Rhus coriaria
L.) leaves and fruits were investigated in the present study. Pomegranate fruits cv. “Mollar de Elche”, at an optimum stage of ripening were from an orchard located in Acireale (Italy). Sumac fruits and leaves were collected in Buccheri (Siracusa, Italy), a typical production area of this crop. After harvesting, all fruits and leaves were immediately transferred to the CRA-ACM laboratories (Acireale, Italy). Then, all plant portions were cut into pieces and oven dried at 40 °C up to constant weight, and then powdered in a homegrinder (Mx Type A505, Moulinex, Ecully Cedex, France). The dried powder of sumac fruits was extracted for 8 h with n
-hexane (VWR Chemicals, Milan, Italy) to remove lipid components using a Soxhlet apparatus. The defatted sumac fruits were put in an oven at 50 °C for 8 h to remove the residue of n
-hexane. Afterwards, the powder was extracted with the extracting solutions. All extracts were obtained from grinded tissues with water or water/ethanol (95%, food grade, Fichera, S. Venerina, Italy) solutions with a ratio of 1:10 (w
). From both pomegranate and sumac plant materials, four kind of hydroalcoholic extracts were tested: (1) 50% ethanol/water (1:1) (v
); (2) 80% ethanol/water (4:1); (3) a concentrated aqueous extract obtained from 80% ethanol/water mixture after evaporation of ethanol by using a rotary evaporator (Rotavapor RE111, Büchi, Cornaredo, Italy) under vacuum at 40 °C; and finally; (4) a water extract obtained using distilled water at 40 °C (Table 1
). The extracting solutions were supplemented with 1% citric acid (Sigma-Aldrich, Milan, Italy) except for the solution used for the preparation of concentrated extracts (80% ethanol/water (4:1)). For this latter extract a 0.2% citric acid solution was utilized in order to have in the final extract (after the evaporation of ethanol) a concentration of approximately 1%. The solutions were left in an orbital shaker (711D model, Tecnolab, Messina, Italy) for 24 h. Then, the solutions were filtered through Miracloth paper (Calbiochem, Vimodrone, Italy) and through a 0.45 μm membrane filter (Albet, Barcelona, Spain) for the subsequent analyses.
3.2. Determination of Total Anthocyanins and Total Phenolic Content of the Plant Extracts
The total anthocyanins in the extracts were assayed by the pH differential method [42
]. The absorbance values of appropriately diluted extracts were measured at 520 nm by a UV-Vis spectrophotometer (Varian Cary 100 Scan, Palo Alto, CA, USA). The total anthocyanins was expressed as mg of cyanidin 3-glucoside equivalents (CGE)/kg of extract. The total phenolic content in the extracts was determined according to the Folin–Ciocalteu (FC) colorimetric method [43
]. The extracts were mixed with 5 mL of FC commercial reagent (previously diluted with water, 1:10 v
) and 4 mL of a 7.5% sodium carbonate solution. The mixture was stirred for 2 h at room temperature away from strong light. The absorbance of the resulting blue solution was measured spectrophotometrically at 765 nm and the total phenolic content was expressed as g of gallic acid equivalents (GAE)/kg of extract.
3.3. UPLC-PDA-ESI/MSn Analyses
For identification of anthocyanins, PGE-C and SUF-C extracts were loaded onto a C18 Bond Elut SPE cartridges (Varian Inc., Palo Alto, CA, USA) pre-washed in methanol and then pre-equilibrated in water. Anthocyanins were adsorbed onto the C18 Bond Elut column, while other soluble compounds were removed by washing the cartridges with water. Then anthocyanins were eluted with acidified methanol (containing 1% of formic acid). The acidified methanol solutions were evaporated to dryness, and then the dry fractions were re-dissolved in a 7% formic acid aqueous solution. Samples were filtered through a 0.45 μm membrane filter (Albet, Barcelona, Spain) and injected into the UPLC-MSn chromatographic system (see below) for identification of individual anthocyanins. The standards of cyanidin 3-glucoside, cyanidin 3,5-diglucoside, delphinidin 3-glucoside, delphinidin 3,5-diglucoside, pelargonidin 3-glucoside, pelargonidin 3,5-diglucoside, and myricetin 3-rhamnoside were purchased from Extrasynthese (Genay, France). Gallic acid, quercetin 3-glucoside, ellagic acid, and punicalagin were purchased from (Sigma-Aldrich). All other chemicals were of analytical grade (Sigma-Aldrich). Solvents for chromatography were HPLC grade (Merck KGaA, Darmstadt, Germany).
For identification of non-anthocyanins phenolic compounds, a sample of PGE-C, SUF-C and SUL-C extracts was diluted in mobile phase solvent A (water containing 0.3% of formic acid), filtered through a 0.45 μm filter, and injected directly into the column.
Separations of anthocyanins and non-anthocyanins phenolic compounds were conducted on an Onyx Monolithic C18 column (100 × 3.0 mm i.d., monolithic particle size; Phenomenex, Torrance, CA, USA), using an Ultra Fast HPLC system coupled to a photodiode array (PDA) detector and a Finnigan LXQ ion trap equipped with an electrospray ionization (ESI) interface in a series configuration (Thermo Electron, San Jose, CA, USA). Two different binary gradients were used. A binary gradient composed of water containing 7% of formic acid (solvent A) and methanol (solvent B) was used for the separation of anthocyanins. The gradient was run as follows: from 5% to 40% of B in 20 min, isocratic for 7 min, followed by re-equilibrating the column to initial conditions. A binary gradient composed of water containing 0.3% formic acid (solvent A) and acetonitrile containing 0.3% formic acid (solvent B) was used for the separation of phenolic compounds. The gradient was run as follows: 0 min, 5% B, 10 min, 2% B, 50 min, 28% B, 60 min, 43% B, isocratic for 20 min, followed by re-equilibrating the column to initial conditions. The flow rate was 300 μL/min, the column temperature was maintained at 30 °C and the injection volume was 20 μL, both for anthocyanins and phenolics analysis. Chromatograms were recorded at 280, 320, 378 and 520 nm.
Operating parameters of the mass spectrometer were the same for both anthocyanins and phenolics analysis: spray voltage 5.00 kV, capillary temperature 275 °C, capillary voltage 19 V. A sheath gas flow rate of 30 arb (arbitrary units) was applied, and the auxiliary and sweep gas were set at 15 and 4 arb, respectively. Preliminary positive and negative tunings were carried out with direct injection of diluted solutions of cyanidin 3-glucoside and punicalagin, respectively at a flow rate of 5 μL/min and the voltages of the optical lenses were optimized by using TunePlus Xcalibur v. 2.0.7 software (Thermo Electron, San Jose, CA, USA). MS full-scan acquisition was carried out from m/z 100 to 2000, in both positive and negative mode. Then, chosen peaks were isolated in the ion trap and fragmented by an MSn scan acquisition. The MSn spectra were obtained by using a collision energy of 13%–17% of instrument maximum, operating with an isolation width (m/z) of 1.5. The mass spectrometry data were acquired in the positive ionization mode for anthocyanins and in the negative ionization mode for other phenolic compounds.
The anthocyanins and phenolics were identified by using their retention times (tR), UV-Vis spectra, co-chromatography with standard in several solvent systems, MS and MSn spectral data operating in positive and negative ion mode, respectively. In addition, comparison of the MS data with those of pure standards and/or those reported in literature was performed. The relative composition (%) of the individual anthocyanins and phenolics were calculated from the peak areas of the chromatograms detected at 520 and 320 nm, respectively, using Xcalibur v. 2.0.7 software (Thermo Electron).